1. Field of the Invention
This invention relates to a dry-type integral multilayer analytical element for the quantitative analysis of total cholesterol in a blood sample.
2. Description of the Prior Art
The total cholesterol in blood consists of bound cholesterol and free cholesterol. The free cholesterol occupies only about 25% of the total cholesterol, and the bound cholesterol occupies about 75%. The bound cholesterol is a complex of cholesterol ester and protein. The free cholesterol can be analyzed quantitatively by oxidizing in the presence of cholesterol oxidase and then determining the produced hydrogen peroxide or cholestenone, but the bound cholesterol cannot be analyzed unless it is decomposed to free cholesterol.
Since the complex of cholesterol ester and protein inhibits the action of cholesterol esterase on cholesterol ester, it is necessary to decompose the complex prior to allowing the esterase to act. As such a means, it is proposed to allow polyethylene oxide monoalkylphenyl ether (alkylphenoxypolyethoxyethanol (synonym)) having an ethylene glycol unit of less than 20 to act (U.S. Pat. Nos. 4,275,151, 4,275,152). It is also known to use a surfactant such as polyethylene glycol alkylether together with cholesterol esterase (U.S. Pat. No. 3,925,164).
On the other hand, in clinical chemical assay analyzing blood samples, it is desirable to conduct accurate analysis by using a small amount of the samples. Heretofore, wet analyses using liquid reagents were widely utilized, but it is inferior in rapidity. While, dry analyses, i.e. the clinical assays using an analytical element substantially in a dry state such as a test piece or a multilayer analytical element containing an analytical reagent system, have been developed since the period of nineteen seventies. The dry analyses are superior to the wet analyses in simple operations, rapidity and low cost. Dry type multilayer analytical elements were developed as an analytical means capable of conducting accurate analysis rapidly using a small amount of samples (U.S. Pat. Nos. 3,992,158, 4,292,272, EP 0 162 302 A, etc.). The dry type multilayer analytical element is constructed, for example, by a transparent support, a reagent layer, a light reflecting layer and a spreading layer. The support is made of a thin plastic film or sheet. The reagent layer coated on the support contains a reagent system reacting with the analyte in a liquid sample to color in an optical density corresponding to the amount of the analyte. The light reflecting layer prevents that the light incident to the reagent layer reaches the spreading layer, and thereby, the influence of the colored liquid sample spotted onto the spreading layer is excluded at the time of the optical measurement of the reagent layer. The spreading layer spreads the liquid sample spotted thereto uniformly into an area in proportion to the liquid amount. When a quantitative analysis is conducted using the above dry type analytical element, a definite amount of a liquid sample such as a whole blood sample is spotted to the surface of the spreading layer. The sample spread in the spreading layer permeates the light reflecting layer to reach the reagent layer. The analyte reacts with the reagent to color or to change color. After spotting, the color reaction is allowed to proceed sufficiently by keeping the analytical element at a definite temperature (incubation) for a suitable time, and then, a light is irradiated to the reagent layer from the transparent support side to determine the reflection optical density by measuring the amount of the reflected light in a restricted wave region. The amount of the analyte can be determined by using a calibration curve which has previously been prepared.
In the past, most of blood samples were treated with the removal of erythrocytes, and the blood sera or blood plasmas thus obtained were subjected to analysis, irrespective of wet analysis or dry analysis. Since the removal of erythrocytes is difficult and requires costly equipment, it is preferred to analyze undiluted whole blood samples as is.
In order to analyze a whole blood sample by dry analysis, it is necessary to separate the blood serum or plasma from blood cells in the analytical element. The dry type analytical element disclosed in EP 0 226 465 A satisfies these requirements and is useful for the analysis of a particular component in a whole blood sample. The analytical element contains a first nonfibrous porous layer, a second nonfibrous porous layer and a fibrous porous layer integrally superposed in this order, and the respective layers are laminated closely by an adhesive partially disposed to form microspaces so as not to interfere uniform permeation of liquid. A reagent composition to produce an optical change such as coloring is incorporated in one or more of the above three layers, and the optical change caused by an analyte is detected mainly at the first nonfibrous porous layer. In the multilayer analytical element, the second nonfibrous porous layer and the fibrous porous layer works cooperatively to remove blood cell components from blood sample.
In the above multilayer analytical element having the layers for separating blood cells at the upper part, the hemolysis in the layers for separating blood cells is a great problem. That is, the hemoglobins or the like are released from red blood cells by the hemolysis, and interfere with the measurement of reflected light in a restricted region. Moreover, the hemolysis changes the total cholesterol concentration of blood sera or plasmas. Sometimes, enzyme reaction or coloring reaction is inhibited by the hemolyzed products. Thus, when hemolysis occurs in an analytical element, particularly in a layer for separating blood cells, the hemolysis reduces the accuracy of the total cholesterol analysis.
The aforementioned polyethylene oxide monoalkylphenyl ether is useful for the hydrolysis of cholesterol, but this compound has a great hemolyzability. Moreover, since the compound is a relatively low molecular weight, its diffusibility to other layers is great. Therefore, when the compound is incorporated into a dry type analytical element having a layer for separating blood cells, hemolysis occurs in the analytical element. As a result, the analysis of total cholesterol concentration cannot be conducted accurately.